All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
NF-κB is a family of dimeric transcription factors. The inhibitor of κB (I-κB) retains NE-κB in the cytoplasm and following exposure to extracellular inducers it undergoes phosphorylation, ubiquination and subsequent degradation. This in turn, allows NF-κB to translocate into the nucleus, where it bind a common sequence motif known as the NF-κB site [Ghosh, S. et al. Annual Review of Immunology 16:225-60 (1998)] and stimulates gene expression (for review, see [Senftleben, U. and Karin, M. Crit. Care Med. 30:S18-S26 (2002)].
DNA damage induced by the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) results in a marked increase in NF-κB activity [Weaver K D. et al. J. Neurooncol. 61:187-96 (2003)]. Inhibition of NF-κB activity by a super-repressor strongly enhances the apoptotic potential of the alkylating agent. Therefore, it was suggested that in human tumors the role of NF-κB in anti-apoptotic mechanisms contributes to the high incidence of chemoresistance to alkylating agents [Weaver (2003) ibid.].
Alkylating agents are highly reactive mutagens and carcinogens and their analogous compounds are used to treat human malignancies. They represent a large class of DNA damaging compounds that include drugs like temozolomide, streptozotocin, procarbazine, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and dacarbazine. The lethal and mutagenic effects of these compounds is inhibited by the cellular DNA-repair enzyme O(6)-Methylguanine-DNA-Methyltransferase (MGMT), which transfers the alkyl/methyl adducts from the (O)6 atom of DNA guanine to its own cysteine residues. The guanine is then restored and the MGMT molecule is irreversibly inactivated [Srivenugopal, K S. Biochemistry 35:1328-34 (1996)]. Hence, the repair capability of MGMT is dependent on de novo protein synthesis. MGMT expression varies widely in tumor cells [Citron, M. et al. Cancer Res. 51:4131-4 (1991); Washington, W J. et al. Mech. Ageing Dev. 48:43-52 (1989)]. It has been suggested that hypermethylation of CpG islands within the promoter region is associated with epigenetic inactivation of the MGMT. Several studies demonstrated that tumor cells with MGMT promoter methylation are more sensitive to alkylating agents such as BCNU and temozolomide [Hegi, M E. et al. N. Engl. J. Med. 352:997-1003 (2005); Danam, R P. et al. Int. J. Oncol. 18:1187-93 (2001); Silber, J R. et al. Proc. Natl. Acad. Sci. USA 93:6941-6 (1996)].
On the other hand, over-expression of MGMT in tumors has a protective effect against cell death induced by chlorethylating and methylating agents, in both experimental and clinical settings Inhibition of MGMT activity, using an artificial substrate such as O6-Benzylguanine (BG), sensitizes the tumor cells to the toxic effects of chemotherapeutic alkylating agents [Esteller, M. et al. N. Engl. J. Med. 343:1350-4 (2000)].
These data indicate that MGMT expression is a crucial player in tumor drug resistance and is an ideal target for modulation. Therefore, an understanding of the molecular mechanisms that control MGMT expression may have major clinical implications.
The promoter region of MGMT has been cloned and sequenced [Harris, L C. et al. Nucleic Acids Res. 19:6163-7 (1991)]. The function of the transcription factors glucocorticoid-responsive element (GRE) [Biswas, T. et al. Oncogene 18:525-32 (1999)] and AP-1 [Boldogh, I. et al. Cancer Res. 58:3950-6 (1998)] in the regulation of MGMT have been described. As shown by the present invention, the inventors identified several putative NF-κB binding sites in the MGMT promoter region and analyzed their role in the regulation of MGMT expression. Using electrophoretic mobility shift assay (EMSA) the inventors found that NF-κB binds specifically to the MGMT promoter and showed that transient transfection of HEK293 cells with the NF-κB subunit p65 induced a 55-fold increase in MGMT expression. The relationships between NF-κB and MGMT were further demonstrated in glioma cell lines and human glial tumors. The inventors found a significant correlation between the extent of NF-κB activation and MGMT expression which was independent of MGMT promoter methylation. The inventors further demonstrated that ectopic expression of p65 or high constitutive NF-κB activity has a protective effect against alkylating agents. This protective effect seems to be solely dependent on MGMT. In view of the fact that a large proportion of tumor cells display high constitutive activation of NF-κB (for review, see [Amit, S. et al. Semin. Cancer Biol. 13:15-28 (2003)]), and that such tumors usually exhibit increased resistance to chemotherapy [Baldwin, A S. J. Clin. Invest. 107:241-6 (2001)] these results are of great clinical importance.
It is therefore one object of the invention to provide a nucleic acid sequence derived from the MGMT promoter region, comprising an NF-κB binding site.
Another object of the invention is to provide a modified nucleic acid sequence comprising at least one copy of an MGMT NF-κB binding site of the invention as an NF-κB decoy. The invention further provides compositions, combinations, kits, methods and uses of said decoy molecule for treating immune related disorders. In yet another object, the invention provides the use of the decoy molecules of the invention and any composition and kits thereof for increasing cell sensitivity to therapeutic agents causing DNA damage, in a subject in need thereof.
It yet another object, the invention relates to a screening method for a compound which inhibits NF-κB mediated expression of MGMT and thereby may be used for treating immune related disorders, also by increasing sensitivity to therapeutic agents.
These and other objects of the invention will become apparent as the description proceeds.